Performing Department
Water and Veterinary Products
Non Technical Summary
Per- and Polyfluorinated substances (PFAS) pose a significant health risk to the United States, since these chemicals are persistent, toxic, bio-accumulative, and ubiquitous. PFAS are commonly found in ground and surface waters, which can lead to elevated levels of PFAS in agricultural products. To protect the safety of the nation's food and water supply, more frequent and widespread PFAS testing is necessary. Currently, methods to detect these chemicals are primarily laboratory-based and the rapid methods that exist have significant limitations. Here, we propose a rapid, quantitative, sensitive test for PFAS that will detect many of these chemicals simultaneously. The test will be based on a competitive immunoassay using peroxisome proliferator-activated receptor alpha (PPARα), which binds to PFAS. The immunoassay will be developed on the LightDeck platform, which uses waveguide technology to perform rapid, easy-to-use immunoassays with a fluorescent readout that is digitally processed to provide a numeric result to users. The primary objective of this proposal is the development of an immunoassay based on PPARα binding to PFAS. Assay range and reproducibility will be measured to ensure that the assay meets customer needs. The test platform has already been developed and is used in LightDeck's commercialized MC + CYN Test. The test is expected to have a lower limit well below the actionable regulatory guidelines for these chemicals. This test will be used in agriculture, water utilities, and manufacturing industries to ensure the safety of the US water supply.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
Per- and Polyfluorinated substances (PFAS) pose a significant health risk to the United States, since these chemicals are persistent, toxic, bio-accumulative, and ubiquitous. PFAS are commonly found in ground and surface waters, which can lead to elevated levels of PFAS in agricultural products. To protect the safety of the nation's food and water supply, more frequent and widespread PFAS testing is necessary. Currently, methods to detect these chemicals are primarily laboratory-based and the rapid methods that exist have significant limitations. Here, we will develop a rapid, quantitative, sensitive test for PFAS that will detect many of these chemicals simultaneously. The test will be based on a competitive immunoassay using peroxisome proliferator-activated receptor alpha (PPARα), which binds to PFAS. The immunoassay will be developed on the LightDeck platform, which uses waveguide technology to perform rapid, easy-to-use immunoassays with a fluorescent readout that is digitally processed to provide a numeric result to users. The primary objective is the development of an immunoassay based on PPARα binding to PFAS.The test is expected to have a lower limit well below the actionable regulatory guidelines for these chemicals. This test will be used in agriculture, water utilities, and manufacturing industries to ensure the safety of the US water supply.The goal of this project is the creation of an immunoassay to detect PFAS with the necessary sensitivity and assay range. Here, we plan to use a competitive immunoassay that uses a capture reagent containing a PFAS which competes with the PFAS present in the sample when binding to a detection reagent. In a competitive immunoassay, the antigen present within a sample competes with an assay reagent for binding sites on an antibody or another bioreceptor. The binding of antigen to the bioreceptor is then quantified using a reporter molecule (i.e., fluorophore), whose signal is proportional to the concentration of antigen.Objective 1, Identify and Procure Reagents: In this objective, reagents will be identified and purchased for use in an immunoassay.Objective 2, Perform conjugation chemistries to create the capture and detection reagents used in the immunoassay: In this objective, protein bioconjugates will be generated for use in assay development to develop the capture and detection reagents necessary for the immunoassay.Objective 2.1: Conjugation of PFOA to BSA. These assays will be performed as competitive assays, where a PFAS-protein conjugate is printed on the surface of the waveguide in a microarray as a capture reagent. In this Phase I project, perfluorooctanoic acid (PFOA) will be conjugated to a carrier protein. Carrier proteins, such as bovine serum albumin (BSA) or ovalbumin (OVA), are commonly used for immobilization of small molecule antigens.Objective 2.2: Conjugation of PPARα to a fluorescent dye. In the simplest assay format, PPARα will be directly labeled with the fluorescent dye. In humans and animals, one reason that PFAS are dangerous is that behave as peroxisome proliferator-response elements (PPREs) that can be ligated by PPARα, which demonstrates that there is expected to be good binding of PFAS to PPARα in this immunoassay. PPARα can be used as a detection reagent in a competitive immunoassay if it is labeled with a fluorophore. In this assay format, PPARα serves as the detection bioreceptor, binding to PFAS and allowing for quantification.Objective 2.3: Conjugation of anti-PPARα to a fluorescent dye. In humans and animals, one reason that PFAS are dangerous is that they behave as peroxisome proliferator-response elements (PPREs) that can be ligated by PPARα, which demonstrates that there is expected to be good binding of PFAS to PPARα in this immunoassay. As an alternative to fluorophore labeled PPARα, anti-PPARα antibodies can bind to PPARα to serve as a detection mechanism. Depending on the assay format, either PPARα or anti-PPARα antibodies need to be conjugated to a fluorescent dye for optical readout.Objective 2.4: Characterization of protein bioconjugates. Establishing well-defined characterization methods for protein bioconjugates is important to ensure assay repeatability and reproducibility.Objective 3, Demonstrate and screen competitive immunoassay formats: Formats for the competitive immunoassay will be explored using the protein bioconjugates prepared in Objective 2. Among the assay formats to be screened are a multi-step turn-off competitive, a single-step turn-off competitive, and a turn-off bioreceptor competitive assay. Each of the assay formats will be initially evaluated to determine a final assay format. Assays will be evaluated based on sensitivity, reproducibility, and ease-of-use, with the best performing assay format optimized in Objective 4.Objective 3.1: Multi-Step Turn-Off Competitive Assay. The first assay format is a multi-step turn-off competitive assay. In this format, BSA-PFOA bioconjugates will be immobilized on the assay surface. Sample containing PFAS will then be added to the PPARα detection reagent off-cartridge, before mixing and incubating the sample to allow for interaction between PFAS and PPARα.Objective 3.2: Single-Step Turn-Off Competitive Assay. If the multi-step assay demonstrated in Objective 3.1 demonstrates feasibility, then a condensed version of the assay will be screened.Objective 3.3: Turn-Off Bioreceptor Competitive Assay. In an alternative assay format, the specificity of the bioreceptor PPARα towards PFOA will be leveraged to further simplify the assay. The fluorescence intensity is measured. Rather than relying on a fluorescent dye-labeled antibody for the detection and readout, the PPARα-dye serves as both the detection and reporter reagents.Objective 4, Optimize Best Performing Competitive Immunoassay: The best performing assay format from Objective 3 will be optimized. In Phase I, optimization of cartridges will have the goal of demonstrating feasibility of the assay format and that the assay sensitivity is tunable.Objective 4.1: Demonstrate Tunability of Assay Cartridge Reagents. The best performing assay format tested in Objective 3 will be used moving forward for the final stages of assay development. In this Objective, the assay tunability using reagents on the assay cartridge will be demonstrated.Objective 4.2: Demonstrate Assay Tunability of Assay Detection Reagents. The best performing assay conditions from Objective 4.1, based on sensitivity and reproducibility, will be used in this Objective.Objective 5, Evaluate analytical performance of immunoassay by measuring the limit of detection, limit of blank, and assay range: In this Objective, the analytical performance of the assay cartridges, developed in Objective 4, will be evaluated. The assay will be evaluated for analytical sensitivity (i.e., limit of blank (LOB), limit of detection (LOD), lower limit of quantification (LLOQ), upper limit of quantification (ULOQ), and range), analytical precision, and accuracy. Analytical sensitivity and precision will be evaluated using spiked laboratory water samples, while accuracy will be measured using certified reference standards. The goal is to achieve a LOD of ≤1000 ppt with a stretch goal of a LOD of ≤10 ppt and percent coefficients of variation (%CVs) of ≤20% over the linear dynamic range of the assay. For this Phase I feasibility study, a LOD of hundreds of ppt is acceptable, but the optimized assay developed in Phase II will have a goal LOD of ≤10 ppt.
Project Methods
Objective 1, Identify and Procure Reagents: There are multiple vendors for PPARα and antibodies against, so it will be necessary to determine which reagents are optimal.Objective 2, Perform conjugation chemistries to create the capture and detection reagents used in the immunoassay.Objective 2.1: Conjugation of PFOA to BSA. Conjugation of several antigen molecules to a protein allows for multiple binding sites per protein, while also allowing for direct adsorption of the carrier protein to the assay surface. PFOA was selected as a model PFAS molecule due to its global prevalence, and the availability of a carboxylic acid functional group for chemical reactivity. The carboxylic acid of PFOA can be conjugated to the amine of lysine residues on the carrier protein, forming an amide bond. The carboxylic acid is first activated using a carbodiimide (i.e., 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)), followed by a nucleophilic addition of the amine to generate the amide bond. This conjugation is expected to be straightforward. Additional optimization work is expected to be performed throughout the program to improve assay performance. Conjugation parameters, such as buffer type, pH, ionic strength, PFOA concentration, carrier protein concentration, and carbodiimide concentration, and others will be investigated to optimize assay performance.Objective 2.2: Conjugation of PPARα to a fluorescent dye. Similar conjugation reactions are routinely performed at LightDeck Diagnostics as part of other assay development programs where antibodies and other proteins are conjugated to fluorescent dyes.Objective 2.3: Conjugation of anti-PPARα to a fluorescent dye. Conjugation parameters need to be tailored specifically to each protein to maximize assay sensitivity and reproducibility.Objective 2.4: Characterization of protein bioconjugates. A key characteristic of protein bioconjugates is the degree of labeling (DOL), which is a measure of the number of molecules of small molecule compound (i.e., PFOA or a fluorescent dye) that are covalently bound to each protein molecule.Capture reagents are immobilized onto the assay surface, so accurately knowing the DOL allows for accurate determination of the reagent quantity being deposited onto the surface. The use of amine-reactive fluorophores (e.g., fluorescamine) will be explored for the determination of degree of labeling (DOL) by PFOA or other PFAS species.Objective 3, Demonstrate and screen competitive immunoassay formats.Objective 3.1: Multi-Step Turn-Off Competitive Assay. The solution containing complexed PFAS-PPARα is then added to the cartridge and allowed to interact with the immobilized BSA-PFOA. PFAS in the sample will compete with the BSA-PFOA for the PPARα receptor binding site, such that increasing PFAS sample concentrations result in decreased PPARα binding with the BSA-PFOA. The assay is then washed to remove excess PPARα, and the anti-PPARα-dye detection reagent is added. The anti-PPARα-dye will bind to any PPARα bound to the BSA-PFOA, producing a fluorescent signal proportional to the amount of PPARα bound to the BSA-PFOA. The sample is then washed to remove non-specifically bound anti-PPARα-dye, and the fluorescence intensity of the sample is measured to quantify the PFAS concentration in the analyzed sample. This assay format is the most user-intensive and would require user multiple steps. The multiple wash steps may be unnecessary, as the LightDeck waveguide only illuminates 100 nm of material above the waveguide surface.Objective 3.2: Single-Step Turn-Off Competitive Assay. As with the assay format in Objective 3.1, BSA-PFOA will be immobilized on the assay surface. Unlike in the previous assay format, the PFAS containing sample will now be incubated with both PPARα and anti-PPARα-dye simultaneously. In this single step format, the complex of PPARα-anti-PPARα-dye is formed prior to addition to the cartridge, reducing the steps performed by the user. PFAS in the sample will compete with BSA-PFOA for the PPARα receptor site, such that increasing PFAS sample concentrations result in decreased PPARα binding with the BSA-PFOA. The sample is then added to the assay, washed, and then the fluorescence intensity of the sample is measured.Objective 3.3: Turn-Off Bioreceptor Competitive Assay. BSA-PFOA will be immobilized directly on the assay surface. PFAS containing sample is added to the PPARα-dye detection reagent, mixed, and incubated to allow for interaction between PFAS and PPARα-dye. The PFAS-PPARα-dye complex solution is then added to the cartridge, where PFAS will compete with BSA-PFOA for the PPARα receptor site. After a possible wash step, the fluorescence intensity is measured. Rather than relying on a fluorescent dye-labeled antibody for the detection and readout, the PPARα-dye serves as both the detection and reporter reagents.Objective 4, Optimize Best Performing Competitive Immunoassay: An assay will be considered tunable if the assay sensitivity can be predictably modulated by changes to the assay conditions. The degree to which the assay sensitivity can be modulated will be calculated using design of experiment (DOE) methods and software.Objective 4.1: Demonstrate Tunability of Assay Cartridge Reagents: The composition of reagents contained within the assay cartridge will vary depending on the assay format selected. The key components of the assay cartridge that will be utilized in assay optimization for this Phase I award are detection reagent, immobilization buffers, and internal controls. The effects of detection reagent concentration and DOL will both be screened to determine the impact on assay sensitivity and reproducibility. Immobilization buffers will have composition, pH, and ionic strength varied to minimize background signal and to improve reproducibility. DOE software will be used to design and plan these optimization studies, with both primary and secondary interactions between reagents being explored. Internal control reagents will be explored to serve as appropriate process control checks.Objective 4.2: Demonstrate Assay Tunability of Assay Detection Reagents. The detection solution may be composed of stabilization reagents, PPARα (either dye conjugated or unconjugated), and/or anti-PPARα-dye. The impact of stabilization reagents on assay performance will be evaluated by varying stabilization reagent composition, buffer composition, concentration, and pH. The stabilization reagents will be optimized to reduce background intensity and to improve assay reproducibility. PPARα concentration and DOL (if dye conjugated) will be explored to determine their impact on assay sensitivity and reproducibility. As with PPARα, anti-PPARα concentration and DOL will be varied to determine their impact on assay sensitivity and reproducibility. The best performing combination of detection reagents will then be converted into a dried form to improve reagent stability and minimize user input. Dried reagents can be reconstituted directly using the sample, removing the need to pipette multiple reagents into the assay solution. DOE software will be used to design and plan these optimization studies, with both primary and secondary interactions between reagents being explored.Objective 5, Evaluate analytical performance of immunoassay by measuring the limit of detection, limit of blank, and assay range: Analytical sensitivity and precision will be evaluated using spiked laboratory water samples, while accuracy will be measured using certified reference standards. These reference standards can be purchased from Supelco, AccuStandard, Wellington Labs, and NIST, among others.